Safety, tolerability, pharmacokinetics and pharmacodynamics of milvexian with aspirin and/or clopidogrel in healthy participants

Milvexian, an oral activated Factor XI (FXIa) inhibitor, is in clinical studies where it may be combined with antiplatelet agents, including aspirin and/or clopidogrel, to prevent thromboembolic diseases. This phase I trial assessed safety, pharmacokinetics, and pharmacodynamics of milvexian coadministration with aspirin and/or clopidogrel in healthy participants through 3 drug-drug interaction studies using a 3-period, 3-treatment, crossover design. A total of 113 participants were randomized to receive milvexian (200 mg; twice daily for 5 days) or matched placebo coadministered with once-daily aspirin (325 mg for 5 days) and/or clopidogrel (Day 1: 300 mg; Days 2–5: 75 mg). Milvexian was safe and well tolerated, with and without aspirin and/or clopidogrel. Eight mild bleeding adverse events (AEs) were reported in 5 of 113 participants across various treatment arms. Peak and total exposures of milvexian were similar with or without clopidogrel and/or aspirin. Exposure-dependent prolongation of activated partial thromboplastin time and reduction of FXI clotting activity by milvexian were similar with coadministration of aspirin and/or clopidogrel. Milvexian, with or without coadministration of aspirin and/or clopidogrel, did not affect bleeding time or platelet aggregation. Administration of milvexian alone or with aspirin and/or clopidogrel was safe and well tolerated without increased incidence of AEs, including bleeding. Pharmacokinetic and pharmacodynamic effects of milvexian, including bleeding time, were similar with or without aspirin and/or clopidogrel. ClinicalTrials.gov Identifier: NCT03698513.

The clinical benefits of antithrombotic therapy are well established for the prevention of thromboembolic events in patients with a broad range of existing cardiovascular diseases, such as nonvalvular atrial fibrillation, acute coronary syndrome (ACS), coronary artery disease (CAD), and peripheral artery disease (PAD) 1,2 .Although improvements in the therapeutic index of anticoagulant therapy have been made, dose-dependent bleeding continues to be observed 3 .Therefore, improving the benefit/risk profiles remains a viable goal for anticoagulant drug discovery.
Blood coagulation involves the activation of plasma proteases, their cofactors, and platelets, with 2 distinct coagulation pathways that converge at Factor X 4,5 .One coagulation pathway, the intrinsic pathway, is important in pathological conditions (ie, thrombotic and thromboembolic events), but not for hemostasis 6 .Factor XI (FXI) is a component of the intrinsic pathway and has been proposed to play an important role in maintaining and propagating a formed thrombus 7,8 .Activated FXI (FXIa) enhances the stability of clots and amplifies thrombin generation when coagulation is initiated by tissue factor or the intrinsic pathway 7,8 .Clinical, preclinical, and epidemiologic studies have shown that modulation of FXI may provide a novel mechanism for systemic anticoagulation without increasing the risk of clinically relevant bleeding in a variety of conditions associated with

Safety and tolerability
Milvexian 200 mg twice daily (BID) given for 5 days with and without antiplatelet therapy (clopidogrel 300 mg once daily [QD] on Day 1 then 75 mg QD on Days 2-4 and/or aspirin 325 mg QD) was safe and well tolerated.No deaths or serious AEs occurred during the study (Table 2).Mild bleeding AEs assessed as related to various treatment groups were reported in 5 participants, and no numerical imbalance in the incidence of bleeding was observed when milvexian was coadministered with dual antiplatelet therapy or with aspirin or clopidogrel, separately.Specifically, in Part 1, a single participant reported 2 events of contusion after administration of   www.nature.com/scientificreports/milvexian + aspirin + clopidogrel (Treatment A) and 1 event after placebo + aspirin + clopidogrel (Treatment C), and 1 participant reported 1 event of gingival bleeding and 2 events of vessel puncture-site bruise after administration of placebo + aspirin + clopidogrel (Treatment C).In Part 2, a single participant reported 1 event of contusion after administration of placebo + clopidogrel (Treatment E).In Part 3, a single participant reported 1 event of anal fissure hemorrhage after administration of milvexian alone (Treatment G), and 1 participant reported 1 event of epistaxis after administration of placebo + aspirin (Treatment H).No notable changes in electrocardiogram (ECG), vital signs, or physical examination results were observed during the study (Supplementary Tables 1-3).

Pharmacokinetics
Impact of aspirin and/or clopidogrel on milvexian After coadministration of aspirin and clopidogrel with milvexian (Treatment A), the maximum observed plasma concentration (C max ) and area under the plasma concentration-time curve from time 0 to time of the last quantifiable concentration (AUC (TAU) ) of milvexian were reduced by 17% (geometric mean ratio [GMR; 90% confidence interval (CI)]: 0.827 [0.750, 0.911]) and 15% (GMR [90% CI]: 0.851 [0.781, 0.927]), respectively, on Day 1, but were not changed on Day 5 compared with milvexian administered alone (Treatment B; Fig. 1).The time of maximum observed concentration (T max ) and elimination half-life (T 1/2 ) of milvexian coadministered with aspirin and clopidogrel (Treatment A) were similar to those after milvexian was administered alone (Treatment B; Supplementary Table 4).Mean peak concentration of milvexian across Treatments A and B occurred at a median T max of 3 to 4 h, and mean (standard deviation [SD]) T 1/2 after the final dose of milvexian was 12.2 (2.46) and 11.4 (1.99) h, respectively.After coadministration of clopidogrel with milvexian (Treatment F), the C max , AUC (TAU), T max and T 1/2 of milvexian were similar compared with milvexian administered alone (Treatment D; Fig. 1, Supplementary Table 4).After coadministration of aspirin with milvexian (Treatment I), the C max , AUC (TAU), T max , and T 1/2 of milvexian were also similar compared with milvexian administered alone (Treatment G).

Impact of milvexian on clopidogrel
After coadministration of clopidogrel with milvexian (Treatment F), the C max of clopidogrel was reduced 9.1% (GMR [90% CI]: 0.909 [0.766, 1.078]) on Day 1 but was similar compared with treatment with clopidogrel alone (Treatment E) on Day 5; AUC (TAU) of clopidogrel with milvexian (Treatment F) on Days 1 and 5 were similar compared with clopidogrel alone (Treatment E; Fig. 2a and b).The C max of clopidogrel acid after Treatment F was similar compared with clopidogrel alone (Treatment E) on Day 1, but reduced 14% (GMR [90% CI]: 0.865 [0.788, 0.950]) on Day 5; AUC (TAU) of clopidogrel acid after Treatment F on Days 1 and 5 were similar compared with clopidogrel alone (Treatment E).When clopidogrel was coadministered with milvexian (Treatment F), median T max and mean T 1/2 of both clopidogrel and clopidogrel acid were generally comparable to the corresponding values observed with clopidogrel alone (Treatment E; Supplementary Table 5).Median T max ranged from 1 to 1.5 h on Day 1 and 0.5 to 1 h on Day 5. Mean T 1/2 values of approximately 1 to 2 h were observed for clopidogrel administered with (Treatment F) and without (Treatment E) milvexian on Days 1 and 5. Mean T 1/2 values for clopidogrel acid were approximately 8 h in both treatments (Treatments F and E) on Days 1 and 5.
Under additional influence by aspirin (coadministration of aspirin and clopidogrel with milvexian; Treatment A), the C max of clopidogrel was reduced 14% (GMR [90% CI]: 0.861 [0.745, 0.995]) and 10% (GMR [90% CI]: 0.904 [0.794, 1.031]) on Days 1 and 5, respectively, while AUC (TAU) was similar on Day 1, but increased 8.7% (GMR [90% CI]: 1.087 [0.894, 1.321]) on Day 5 compared with aspirin and clopidogrel without milvexian (Treatment C; Fig. 2); the C max and AUC (TAU) of clopidogrel acid after Treatment A were similar on Days 1 and 5 compared with aspirin and clopidogrel without milvexian (Treatment C).When clopidogrel was coadministered with aspirin and milvexian (Treatment A), median T max and mean T 1/2 of both clopidogrel and clopidogrel acid were generally comparable to the corresponding values observed with aspirin and clopidogrel without milvexian (Treatment C; Supplementary Table 5).

Impact of milvexian on aspirin and/or clopidogrel on bleeding time and platelet aggregation
Mean bleeding time at baseline ranged from 3.78 to 4.56 min across treatment groups.Administration of milvexian alone (Treatments B, D, and G) did not increase bleeding times; mean bleeding times ranged from 4.16 to 5.08 min at 4 h following milvexian administration on Days 1 and 5 (Fig. 5a).Coadministration of aspirin and/or Platelet aggregation induced by adenosine diphosphate (ADP) or collagen was changed by < 10% compared with baseline following administration of milvexian alone (Treatment B, D, and G) on Days 1 and 5 (Fig. 5b, c).Although a decrease in arachidonic acid (AA)-induced platelet aggregation was observed following administration of milvexian alone (Treatment B, D, and G), this was not consistent and did not correlate with milvexian concentration (Fig. 5d).Coadministration of aspirin and clopidogrel with or without milvexian (Treatments A and C, respectively) resulted in ~ 50%, 60%, and 95% inhibition of ADP-, collagen-, and AA-induced aggregation, respectively, compared with baseline.Administration of clopidogrel alone (Treatment E) resulted in ~ 50% inhibition of ADP-induced aggregation and ~ 60% inhibition of AA-induced aggregation with little effect (< 10%) on collagen-induced aggregation compared with baseline.Administration of aspirin alone (Treatment H) resulted in ~ 15% inhibition of ADP-induced aggregation, ~ 25% inhibition of collagen-induced platelet aggregation, and ~ 95% inhibition of AA-induced platelet aggregation compared with baseline.

Discussion
Despite clear evidence that the combination of anticoagulant and antiplatelet therapy reduces the incidence of thrombotic events in patients with cardiovascular disease (eg, ACS, CAD, or PAD), it is also associated with an increase in the risk of bleeding-related AEs [27][28][29][30] .Therefore, it is important to fully characterize the potential DDI of new anticoagulant agents with the current guideline-recommended antiplatelet drugs in these patient populations.Aspirin and clopidogrel are some of the most commonly recommended antiplatelet therapies for treatment of and to reduce the risk of thrombotic events in patients with coronary disease and non-cardioembolic stroke [14][15][16][17] .This phase I study evaluated the potential DDI between milvexian and aspirin and/or clopidogrel in healthy participants.The findings demonstrate that there is no evidence of increased risk of serious or nonserious AEs, including serious bleeding events, associated with the administration of multiple doses of milvexian with dual antiplatelet therapy (aspirin and clopidogrel) or single antiplatelet therapy (aspirin or clopidogrel) or significant changes in PK parameters of milvexian in healthy participants.The percentage of participants reporting AEs was generally consistent across treatments in each part of the study with a slightly higher number of AEs reported after coadministration of placebo and aspirin in Part 3 (23.7%versus 13.5% to 19.4% across all other treatments).Further, the number of participants reporting bleeding AEs was similar across treatments; there was no numerical imbalance in the incidence of bleeding AEs and all bleeding AEs were mild.The results showed no evidence of an increased risk of AEs overall, including bleeding, associated with the administration of multiple doses of milvexian, with or without dual antiplatelet therapy.
Regarding PK, coadministration of aspirin and/or clopidogrel with milvexian did not affect the median T max and mean T 1/2 of milvexian, aspirin (acetylsalicylic acid) and its metabolite (salicylic acid), and clopidogrel and its metabolite (clopidogrel acid).Coadministration of aspirin and clopidogrel with milvexian did not alter the C max and AUC (TAU) of milvexian on Day 5, although slight reductions were observed on Day 1 (C max , 17%; AUC [TAU] , 15%; both were not statistically significant), compared with milvexian administered alone.Coadministration of milvexian with clopidogrel did not alter the C max and AUC (TAU) of clopidogrel and its metabolite.However, coadministration of milvexian with aspirin increased the C max and AUC (TAU) of acetylsalicylic acid on Day 5, compared with treatment with aspirin alone, whereas coadministration of milvexian with aspirin had no effect on the salicylic acid metabolite.Potential explanations for increased exposure in acetylsalicylic acid include: an increase in bioavailability due to disintegration of a tablet formulation of aspirin 31 , period effect, and gastric emptying rates 32,33 .Nevertheless, it should be noted that this increase in acetylsalicylic acid exposure was not expected to be clinically relevant by investigators given the small magnitude of increase.
Prolongation of aPTT and reduction of FXIc were observed after administration of milvexian with greater effects observed at higher concentrations.These observations are consistent with milvexian's mechanism of action and previous observations in phase I studies [18][19][20] and the in vitro studies and in vivo evaluation in experimental thrombosis in rabbits 34 .No additional prolongation of aPTT or reduction in FXIc were observed when milvexian was coadministered with aspirin or clopidogrel compared with administration of milvexian alone; this lack of effect on coagulation tests is expected based on the mechanism of action of clopidogrel and aspirin observed in other studies 2,21,35 .Additionally, these results are similar to previous observation from a phase I study of SHR2285 (ClinicalTrials.govIdentifier: NCT04945616), a small-molecule FXIa inhibitor, which showed that when combined with aspirin, clopidogrel, or ticagrelor, SHR2285 was safe and well tolerated with significant effects on aPTT and FXI activity and no evidence of an increased risk of bleeding 36 .Furthermore, the current study found no effects on platelet aggregation beyond the effects of aspirin and/or clopidogrel alone with coadministration of milvexian.Platelet aggregation was not affected by administration of milvexian alone.These observations are consistent with the mechanism of action of milvexian, which selectively inhibits FXIa and acts as an anticoagulant 13,18,19,34 .
Milvexian has been investigated in 2 phase II studies.In the AXIOMATIC-TKR study of patients who underwent knee arthroplasty, postoperative treatment with milvexian reduced the incidence of venous thromboembolism in a dose-dependent manner (ranging from 25 mg QD to 200 mg BID) and was associated with a low risk of bleeding 37 .The safety and efficacy of milvexian were also investigated in the phase II AXIOMATIC-SSP study of patients with acute ischemic stroke or high risk transient ischemic attack and high risk of recurrent stroke on a background of aspirin and clopidogrel 38 ; milvexian (25 mg QD or 25, 50, 100, 200 mg BID) did not produce a statistically significant dose response for the composite outcome of symptomatic ischemic stroke or covert brain infarction and did not increase symptomatic intracranial or fatal bleeding compared with placebo 39 .
The current study showed no evidence of a safety impact with no increased risk of bleeding and limited impact on PK and PD profiles.A key strength of this study is that overall it is a relatively large, comprehensive phase I study design controlling for intra-individual variability across all potential interactions among the aforementioned drugs.A potential limitation of the study is the small sample sizes in each part of the study.Additional measurements of the concentrations of aspirin and/or clopidogrel and their metabolites are warranted for future studies to confirm potential DDIs of milvexian with aspirin and/or clopidogrel as well as to confirm the safety findings of the current study.Another potential limitation is the generalizability of the study findings as the study population included healthy individuals who were relatively young, thus, these results may not fully extend to patients with cardiovascular diseases or individuals of older age.

Conclusions
This phase I DDI study in healthy adults demonstrated that administration of milvexian alone or in combination with aspirin and/or clopidogrel was safe and well tolerated and was not associated with an increased incidence of AEs, including bleeding.PK parameters and PD effects of milvexian were similar when milvexian was administered alone or in combination with aspirin and/or clopidogrel.Likewise, PK parameters and PD effects of aspirin and/or clopidogrel were generally similar when aspirin and/or clopidogrel were administered alone or in combination with milvexian.Lack of effect of milvexian on bleeding time may have promising safety implications for its potential use as an add-on to aspirin and/or clopidogrel.The results obtained in this study will help to inform the future clinical development of milvexian. https://doi.org/10.1038/s41598-024-67182-8 https://doi.org/10.1038/s41598-024-67182-8www.nature.com/scientificreports/PharmacodynamicsImpact of aspirin and/or clopidogrel on milvexian aPTT and FXIcExposure-dependent prolongation of activated partial thromboplastin time (aPTT) and reduction of Factor XI clotting activity (FXIc) were observed with milvexian administration, and effects were similar when milvexian was administered alone (Treatments B, D, and G) or in combination with aspirin and/or clopidogrel (Treatments A, F, and I; Fig.4a, b).The maximum mean aPTT value ranged from 68.3 to 72.2 s at 16 h following milvexian administration alone (Treatments B, D, and G) or in combination with aspirin and/or clopidogrel (Treatments A, F, and I) on Day 1 and returned to baseline by 72 h postdose on Day 5 (Fig.4a).Similarly, the minimum mean FXIc ranged from 26.7% to 32.5% at 16 h following milvexian administration alone (Treatments B, D, and G) or with aspirin and/or clopidogrel (Treatments A, F, and I) on Day 1 and returned to baseline by 72 h postdose on Day 5 (Fig.4b).

Figure 1 .Figure 2 .Figure 3 .
Figure 1.Effect of aspirin and/or clopidogrel on milvexian PK parameters on Days 1 and 5. (a) Mean (± SD) milvexian plasma concentration versus time profile.* (b) GMR (90% CI) of milvexian C max and AUC (TAU) .AUC (TAU) , area under the plasma concentration-time curve from time 0 to time of last quantifiable concentration; BID, twice daily; CI, confidence interval; C max , maximum observed concentration; GMR, geometric mean ratio; PK, pharmacokinetics; QD, once daily; SD, standard deviation.* Individual plots of the curves in panel a on Days 1 and 5 can be found in Supplementary Fig. 1. † Milvexian 200 mg BID on Days 1 to 5. ‡ Aspirin 325 mg QD on Days 1 to 5. § Clopidogrel 300 mg QD on Day 1 then 75 mg QD on Days 2 to 5.
†Aspirin 325 mg QD on Days 1 to 5. ‡ Clopidogrel 300 mg QD on Day 1 then 75 mg QD on Days 2 to 5.

Table 2 .
Summary Safety and AEs by Treatment.AE, adverse event; BID, twice daily; QD, once daily.*Milvexian 200 mg BID on Days 1 to 5.
† Aspirin 325 mg QD on Days 1 to 5. ‡ Clopidogrel 300 mg QD on Day 1 then 75 mg QD on Days 2 to 5. § Participant discontinued study treatment due to a nonrelated AE of pulpitis dental after milvexian.‖ Participant discontinued study treatment due to a nonrelated AE of hypotension after placebo + clopidogrel.¶ Participant discontinued study treatment due to a related bleeding AE of anal fissure hemorrhage after milvexian.# Participant reported gingival bleeding and 2 events of vessel puncture-site bruise after placebo + aspirin + clopidogrel.**1 participant reported 2 events of contusion after milvexian + aspirin + clopidogrel in Period 1 and 1 event after placebo + aspirin + clopidogrel.† † Participant reported contusion after placebo + clopidogrel.‡ ‡ Participant reported epistaxis after placebo + aspirin.